Note: Descriptions are shown in the official language in which they were submitted.
r
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Extracts from the bark of corynanthe species and use thereof as well as
medicaments, dietetic food products and pharmaceutical preparartions
containing said extracts
The present invention relates to extracts from the bark of corynanthe species,
in
particular corynanthe pachyceras, as well as their use for the therapy and
prophylaxis of diseases of the lower urinary tract, sexual dysfunctions,
disorders of
the lipid metabolism, cardiovascular diseases and acute and chronic pain
conditions.
The present invention further relates to medicaments, dietetic food products
and
pharmaceutical preparations containing said extracts.
Benign prostatic hyperplasia (BPH) and the accompanying lower urinary tract
symptoms (LUTS) are by far the most important urologic diseases in male
humans.
Estimations assume that one third of the male humans above age 50 develop LUTS
in the course of their life and that a surgical intervention will be necessary
for 25 % of
them. The percentage of male humans suffering from BPH/LUTS increases to more
than 90 % until the 9th decade of their life. In the Federal Republic of
Germany about
4 million patients are treated against these complex of symptoms a year. In
view of
the growing expectancy of life and the increased health awareness, a further
increase of the disease frequency has to be bargained for in the future (R. B.
Moreland et al., J. Pharmacol. Exp. Ther. 2004, 308, 797).
Despite the large clinical importance of BPH/LUTS the aetiology and the
pathogenesis of this disease have not been elucidated to a large extent. In
addition
to an increased age, an endogenous production of androgens is an important
precondition for the development of BPH. In general, BPH is thus considered as
an
endocrinopathy of senescent men, which develops as a consequence of the
hormonal reorganization with growing age.
Under a histologic point of view, BPH is the benign growth of the epithelial
and
stromal parts of the prostate. Due to the localization of the prostate at the
urethra
near the exit of the bladder, urinary obstructions which are accompanied by
symptoms such as pollacisuria and nycturia as well as an incomplete and
delayed
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micturition, occur due to an enlargement of the organ. In an advanced stage
renal
insufficiency and uraemia may occur as a result of the urinary stasis. In
addition, due
to stasis of secretions and urinary retentions, an abacterial prostatitis,
congestions
and recurring urinary tract infections develop, which are responsible for
irritative
micturition disorders in addition to obstructive complaints.
In addition to a static component which is due to an enlargement of the
prostate and
the mechanical micturition disorders caused thereby, a dynamic component
appears
to be invoived in BPH/LUTS which is elucidated by an increased tonus of the
smooth
muscles. The extent of involvement of both mechanisms may vary largely from
patient to patient. This explains that there is only a marginal correlation
between the
size of the prostate and the severity of the symptoms (C. G. Roehrborn and D.
A.
Schwinn, J. Urol. 2004, 171, 1029). Basically similar changes of the tonus of
the
smooth muscles are also involved in other diseases of the lower urinary tract,
also in
female humans, such as stress incontinence, urge incontinence and disorders of
emptying the bladder.
Whereas the term BPH is reserved for the histologic or macroscopic diagnosis
of
hyperplasia of the prostatic gland, the accompanying LUTS such as urge to
urinate,
pollacisuria and nycturia as well as an incomplete and delayed micturition
prevail for
the patients. The variety of treatment alternatives for BPH ranges from an
wait-and-
see attitude ("watchful waiting") to an open prostatectomy. Due to its
effectiveness,
the transurethral resection of the prostate, which about 33,500 male humans
are
subjected to per year in Germany, is considered to be the Golden Standard of
surgical methods. However, the considerable risk of morbidity and mortality of
invasive treatment methods is not acceptable for many patients, in particular
patients
with a lower severity of BPH, which explains the increasing importance of
therapy
approaches with medicaments. In addition to phytopharmaceuticals which are
frequently used in case of less severe disease stages, the variety of
medicaments
includes primarily a,-antagonists and 5-a-reductase inhibitors (E. Koch,
Planta Med.
67, 489 - 500 (2001)).
The use of a,-antagonists is based on the view that the dynamic component of
BPH/LUTS is caused by an increased tonus of the smooth prostatic muscles which
is
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mediated by an elevated release of noradrenaline from sympathic neurons. Today
it
is generally acknowledged that predominantly aIA-adrenoceptors and a1D-
adrenoceptors are expressed in the prostate. The results of clinical studies
show that
a-receptor blockers produce a clinically significant improvement of the
symptoms and
of the maximum urinary flow. A particular advantage of a-receptor blockers is
their
rapid onset of action. However, there is currently no convincing evidence that
they
impede the further enlargement of the prostate. The side effects of a-receptor
blockers include dizziness, headaches, weakness, orthostatic dysregulation,
rhinitis
and sexual dysfunction (retrograde ejaculations) which are prevalently caused
by the
action of a-receptor blockers in the CNS and the cardiovascular system. The
development of subtype specific a-receptor blockers which predominantly
inhibit
alA-receptors and alD-receptors (for example tamsulosin), intends to reduce
the
frequency of occurrence and the severity of side effects. However, the non-
selective
a,-antagonist alfuzosin interestingly exhibits a similarly favourable profile
of side
effects as tamsulosin which is generally described as a uroseiective a1-
receptor
blocker. Besides the pharmacodynamic actions the pharmacokinetic properties
appear to significantly contribute to the uroselectivity. Hence, for example,
effects on
the blood pressure can be prevented by means of a slow dosage or sustained
release formulations. Moreover, also alA-receptors are involved in the control
of the
blood pressure in addition to a1B-receptors (R. B. Moreland et al., J.
Pharmacol. Exp.
Ther. 308, 797, 2004; C. G. Roehrborn and D. G. Schwinn, J. Urol. 171, 1029,
2004)
which limits the possibilities to develop uroselective a-receptor blockers.
In general, a BPH develops in the presence of biologically effective male sex
hormones only. A BPH has virtually never been observed in men which had to
undergo castration before the age of 40 or in which no formation of androgens
or an
insufficient formation of androgens only occurs in the gonads due to a
hypofunction
of the hypophysis. Likewise, the normal development of the prostate and the
development of a BPH does not occur in the case of a hereditary defect or in
the
absence of androgenic receptors (for example testicular feminization). The
biologically most important androgen is dihydrotestosterone (DHT) which is
formed
locally from testosterone under the influence of 5-a-reductase. Since DHT
predominantly stimulates the epithelial components of BPH, a standstill of the
growth
or an atrophy of the glandular component can be achieved by inhibiting 5-a-
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reductase. However, the stromal component of BPH is virtually not effected at
all.
For example, the uptake of the 5-a-reductase inhibitor finasteride leads to a
reduction of the prostatic DHT concentration of up to 85 %, whereas the
average
reduction of the prostate size amounts to about 20 % only and additionally
requires a
period of up to 12 months. This effect is of clinical relevance essentially
only in cases
where the volume of the prostate is higher than 40 ml at the beginning of the
therapy.
Although androgens play a central role in the normal development and function
of
the prostate as well as in the development of a BPH, male sexual hormones
alone
are not sufficient to elicit the growth of prostatic cells. Numerous
experimental
investigations show that the growth stimulating effects of androgens in vivo
are
mediated by the local synthesis of growth factors and that dysfunctions in the
paracrine and autocrine mechanisms of the growth control within the epithelial
and
stromal components of the prostate are substantially involved in the
development of
PBH. A variety of growth factors (for example Epidermal Growth Factor [EGF],
Transforming Growth Factor-a [TGF-a], TGF-R, basic Fibroblast Growth Gactor
(bFGF), Keratinocyte Growth Factor [KGF], Nerve Growth Gactor [NGF], Insulin-
like
Growth Factor I [IGF-1] etc.) and their receptors have actually been detected
in the
prostate. Since BPH is very frequently accompanied by inflammatory reactions
which
presumably play an important role in the pathogenesis, Platelet-derived Growth
Factor (PDGF), which is released for example by fibroblasts, thrombocytes and
leucocytes, is probably of particular importance for the proliferation of
prostatic cells
(C. J. Vlahos et al., J. Cell. Biochem. 52, 404 - 413 (1993)).
Growth factors mediate their biological action by binding to specific
receptors at the
surface of the cells, which have an intrinsic tyrosine kinase activity. After
binding the
ligand, the phosphorylation of the tyrosine residues occurs in the
intracellular
receptor domains, which subsequently elicits a cascade of intracellular
reactions.
The stimulation of the protein synthesis and DNA synthesis as well as the
activation
of the cell proliferation are among these reactions. Thus, inhibitors of
receptor
tyrosine kinase are considered to be promising substances for the development
of
new medicaments for the treatment of diseases which are accompanied by an
increased cell proliferation (for example cancer, atherosclerosis, psoriasis)
(A.
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Levitzki and A. Gazit, Science 267, 1782 - 1788 (1995)). However, little
attention has
been paid to this mode of action in the therapy of BPH up to now.
In recent years it has been demonstrated in different epidemiological
investigations
that there is a close correlation between BPH/LUTS and the occurrence of an
erectile dysfunction (ED). For example, in the MSAM-7 study it has been
demonstrated that the prevalence of EP in men without LUTS at an age between
50
and 80 years is about 25 %. This rate increases in patients with severe
symptoms to
more than 80%. Furthermore, the frequency of occurrence of ED is increased by
other simultaneously existing diseases such as hypertension, diabetes,
hypercholesterolemia, angina pectoris and depressions (M. Shabbir et al.,
Curr. Med.
Res. Opin. 20, 603, 2004).
The relaxation of the penis is predominantly maintained by the binding of
noradrenaline to alA-receptors and alB-receptors in the corpus cavernosum. It
is thus
not surprising that an improvement in the sexual function was observed in
patients
with ED by therapy with a-receptor blockers (for example doxazosin and
tamsulosin).
In contrast, erections are predominantly mediated by the vasodilatory effect
of nitric
oxide (NO). NO is released from nitrergic neurons and is further synthesized
by
endothelial cells in the corpus cavernosum and corpus spongiosum. By
stimulating
guanylate cyclase and increasing the synthesis of cGMP, NO causes a relaxation
of
smooth muscle cells. Thereby a combination of a,-antagonistic and a2-
antagonistic
effects is regarded as particularly favourable for the treatment of ED because
the
inhibition of al-receptors directly produces a muscle relaxation and the
inhibition of
presynaptic a2-receptors is accompanied by an increased
release of NO from nitrergic neurons
(http://www.bioportfolio.com/leaddiscovery/mdi002.htm).
The treatment of ED has been revolutionized by the development of sildenafil,
a
PDE5 inhibitor which inhibits the degradation of cGMP. However, sildenafil
causes a
variety of side effects (such as headaches, impaired vision, dyspepsia,
hemodynamic effects). Furthermore, there is evidence that the efficacy
decreases
with an increasing period of treatment (M. Shabbir et al., Curr. Med. Res.
Opin. 20,
603, 2004). Furthermore, sildenafil can not be used by 30 - 50 % of the
patients due
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to the severity of disease and the contraindications. There is currently not a
wide
clinical experience with new PDE5 inhibitors. Also PGE, is an active substance
for
the treatment of ED, however, it has to be injected or administered
intraurethrally. As
a further treatment option apomorphine is available, which acts as a dopamine
agonist in the central nervous system. It is suitable for rather less severe
cases of
ED. Having a generally lower side effect rate (nausea, cardiovascular
effects), the
sublingual administration which is required to avoid a hepatic first pass
metabolization, is regarded as rather disadvantageous (R. B. Moreland et al.,
J.
Pharmacol. Exp. Ther. 2004, 308, 797). Thus, there is still a large demand for
effective medicaments for the treatment of ED, which are low in side effects.
In 1999 a publication which shows that the portion of females with sexual
dysfunction
(about 43 %) is significantly higher than that of male (about 31 %) in the US
(E. 0.
Laumann et al., JAMA 281, 537, 1999), has been recognized among experts with
surprise. In general, sexual dysfunctions in female are classified into four
categories:
lack of sexual desire or sexual aversion, reduced sexual excitability, painful
sexual
intercourse (vaginism, dyspareunia) and orgasmic disorders. The portion of
these
different disorders are 30 % (lack of sexual desire), 20 % (reduced sexual
excitability), 10 to 15 % (painful sexual intercourse) and 10 to 15 %
(orgasmic
disorders), wherein there is a very close relationship between the different
kinds of
sexual dysfunctions.
The regular sexual function in male and female is controlled via a response
cycle
which consists of a mental expectation (sexual desire), effective
vasocongestion
(erection in men, swelling of the clitoris and vaginal lubrication in women),
orgasm
and finally resolution. This overall course of events depends on a balanced
equilibrium between the parasympathetic and sympathetic nervous system.
Thereby,
the vasocongestion of the sexual organs is of central importance. Since there
is an
analogy between the penis and the clitoris with respect to the anatomic
structure and
the innervation of the corpora cavernosa, it is to be anticipated that the
same
pharmacological mechanisms, which are efficient in the treatment of erectile
dysfunction, can also be employed in the treatment of female sexual disorders
and
particularly reduced sexual irritability.
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Therefore, it is the object underlying the present invention to provide
medicaments
which positively effect both the dynamic and static component of BPH by
inhibiting
a1-adrenoceptors and a2-adrenoceptors and by inhibiting the growth factor
mediated
proliferation of epithelial cells and stromal cells and, thus, enable the
comprehensive
treatment of the BPH syndrome, LUTS, ED and other sexual dysfunctions in men
and women as well as hypercholesterolemia, functional disorders of the
bladder,
cardiovascular diseases and pain conditions.
According to the present invention, this object is solved by the use of
extracts from
the bark of corynanthe species, preferably corynanthe pachyceras for the
therapy
and prophylaxis of diseases of the lower urinary tract in men and women (for
example benign prostatic hyperplasia, LUTS, carcinoma of the prostate,
disorders in
emptying the bladder, urinary retention, stress incontinence and urge
incontinence),
sexual disorders in men and women (such as impotence, erectile dysfunction,
premature ejaculation, libido disorders, frigidity or anorgasmy), disorders of
the lipid
metabolism (for example hypercholesterolemia, hyperlipidemia,
hypertriglycerinemia), cardiovascular diseases (for example endothelial
dysfunction,
hypertonia, arteriosclerosis or restenosis after vasodilatation or bypass
surgery) and
acute and chronic pain conditions such as migraine, neuropathic pains (for
example
in case of diabetes), phantom limb pain, allodynia, pains after tissue
injuries or in
case of inflammations (for example postherpetic neuralgia).
Further subjects of the present invention are extracts from the bark of
corynanthe
species, preferably from the bark of corynanthe pachyceras, which have a
balanced
ratio of effective components, as well as medicaments and food products for
the
therapy and prophylaxis of diseases of the lower urinary tract, sexual
disorders,
disorders of the lipid metabolism, cardiovascular diseases and acute and
chronic
pain conditions, which are characterized by a content of an extract according
to the
present invention, as well as a pharmaceutical preparation as an oral,
parenteral or
topicalal administration form. As used herein, the term "food products"
particularly
refers to dietetic food products, dietary supplement products as well as
medical food
and dietary supplements.
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Corynanthe pachyceras (rubiaceae) is a tree having a height of 15 - 20 m and a
trunk diameter of up to 60 cm in the evergreen tropical rain forest in Western
Africa
(Sierra Leone to Zaire). The wood is prevalently used for building purposes,
but also
for the manufacture of mortars and combs. The dried bark of the trunk is
extensively
used in traditional medicine. The bark is chewed against colds and used as a
decoction in case of leprosy, gastric complaints, diarrhea or cardiac and
renal
complaints. The bark is used in the form of teas as an antipyretic agent in
case of
malaria and as an aphrodisiac and a wake-promoting agent.
The bark of corynanthe pachyceras contains about 6 % of indolalkaloids which
are
assigned to the group of corynantheine alkaloids (for example
dihydrocorynanteine,
corynantheine, corynantheidine) or yohimbine alkaloids (for example
corynanthine,
a-yohimbine). For the particular corynanthe alkaloids the focus lies on their
a-
adrenoceptor antagonistic activity. Furthermore, a leishmanicidal activity at
a
moderate cytotoxicity for mammal cells and plasmodium falciparum is described
(D.
Staerk et al., Planta Med. 2000, 66, 531, 2000).
\ N H C~NN H
N H H
H H
H3C0 OH H3C0 OH
O O
Corynanthine a-Yohimbine
' N ~~H ~ I ' \ N Hir
'
H' ~~ CH3 ; H $ OCH
H H - H H
H3C0 0 H H3C0 H
O O
Corynantheine Dihydrocorynantheine
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I \ \ N H
H' OCH3
H H
H3c0 H
O
Corynantheidine
A patent specification from the year 1971 claims antihypertensive and sedative
effects of an aqueous dry extract from the bark of corynanthe pachyceras
(BE758049, Omnium Chimique SA, 1971).
We now have surprisingly observed that alcoholic or ketonic, preferably
ethanolic-
aqueous extracts from the bark of corynanthe species, in particular corynanthe
pachyceras exhibit a variety of further biological effects such as cell
proliferation
inhibiting properties, endothelium dependent vasorelexing properties,
cholesterol
lowering properties, analgetic and antioxidative properties, in addition to al-
adrenoceptor antagonistic and a2-adrenoceptor antagonistic effects. These
different
activities suggest the therapeutic use of these extracts against various
disease
conditions. BPH, LUTS, sexual dysfunction in men and women, functional
disorders
of the bladder, hypercholesterolemia, arteriosclerosis, endothelial
dysfunctions and
pain conditions are among this diseases. The efficacy of the extracts
according to
the present invention against these indications is supported by the following
pharmacologic investigations. In these investigations it has turned out to be
essential
for the effect of the extracts according to the present invention that the
extracts have
to contain polyphenols as active ingredients in addition to alkaloids.
Thereby, the
term "polyphenols" refers to aromatic compounds having at least two hydroxyl
groups
which may be present in the monomeric, oligomeric or polymeric form. Extracts
containing both groups of compounds are clearly superior to the isolated
ingredients
from corynanthe pachyceras with respect to their overall effect.
The extracts according to the present invention from the bark of corynanthe
species,
which have a content of polyphenols and alkaloids, can be obtained according
to the
following method:
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(a) extracting dried and ground bark of corynanthe species with an organic
solvent
or water or a mixture of one or more organic solvents and/or water at a
temperature between 10 C and 100 C,
(b) separating the extracted plant material from the extract solution, for
example by
filtration,
(c) optionally reextracting the extracted plant material with a solvent
according to
step (a) and separation according to step (b),
(d) combining the extract solutions obtained in steps (b) and (c),
(e) evaporating and drying the combined solution from step (d) to yield the
dry
extract.
Preferred organic solvents in step (a) are alcohols or ketones, wherein the
alcohol is
preferably ethanol. Mixtures of ethanol and water are particularly preferred.
Maceration and percolation may be preferably taken into consideration as the
extraction method in step (a). As a general rule, step (c) is carried out once
and
waving step (c) or a plural performance is possible as well. The drying in
step (e) can
be carried out by methods known per se, such as lyophilization or drying in
vacuum
at room temperature or elevated temperature.
Corynanthe pachyceras is employed as the preferred corynanthe species.
The extracts according to the present invention from the bark of corynanthe
species
contain both polyphenois and alkaloids in a ratio balanced for the intended
use.
Thereby, the content of polyphenols is preferably at least 15 %, particularly
preferred
at least 24 % and the content of alkaloids is preferably at least 8 %,
particularly
preferred at least 12 %. The compounds epicatechine, procyanidin B2 and
procyanidin Cl have been isolated by us from corynanthe pachyceras as typical
polyphenols. However, corynanthe polyphenols are not limited to these three
compounds.
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OH ' OH
a
OH
OH
Epicatechine
OH
HO O OH
\ OH
I 1'''OH OH
OH
HO 0 OH HO OH
I / I
OH
'I''OH \ OH / '.,/OH ()~OH
OH OH
HO O OH HO O .1'I'''OH 11''OH
OH OH
Procyanidin B2 Procyanidin Cl
A determination of the contents of polyphenols was carried out by determining
the
total phenol content according to Folin-Ciocalteu. Additionally or
alternatively, the
contents of epicatechine, procyanidin B2 and procyanidin Cl can also be
determined. The alkaloid contents mentioned are the sum of the contents of the
individual alkaloids corynanthine, a-yohimbine, corynantheine,
dihydrocorynantheine
and corynantheidine.
The extracts and extract fractions according to the present invention may be
administered in the form of droplets, powders, granules, tablets, coated
tablets
(dragees) or capsules, preferably orally. However, a parenteral application in
the
form of an injection solution or a topicalal application in the form of
cremes,
ointments, suppositories, patches or similar preparations is also possible.
For the preparation of tablets the extract is mixed with suitable
pharmaceutically
acceptable adjuvants such as lactose, cellulose, silicon dioxide,
croscarmellose and
magnesium stearate and pressed into tablets which may optionally be provided
with
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a suitable coating made of, for example, hydroxymethylpropylcellulose,
polyethyleneglycol, colorants (such as titanium dioxide, iron oxide) and
talcum.
The extracts according to the present invention may also be filled into
capsules
optionally under the addition of adjuvants such as stabilizers, fillers and
the like.
The dosage is such that 5 to 2000 mg, preferably 10 to 1000 mg and
particularly
preferred 50 to 500 mg extract are administered per day.
The efficacy of the extracts according to the present invention from the bark
of
corynanthe species is supported by the experiments described in the following.
Pharmacological investigations
Assay for a-adrenoceptor binding properties
The test of corynanthe extracts and fractions of corynanthe extracts for
interactions
with a-adrenoceptors was carried out by a receptor binding assay using brain
cell
membranes of rats. For the preparation of cell membranes, male Sprague-Dawley
rats (150 - 250 g) were euthanized in CO2 narcosis and the brains were removed
(without the cerebellum). After the removal of adhering blood and meninges,
the
brains were immediately taken up in ten times their volume made up of ice cold
homogenization buffer (50 mM Tris-HCI, pH 7.4) and homogenized using an ice
cooled glass homogenizor. The cell homogenate was centrifugated for 10 minutes
at
50,000 g (4 C) and the pellet was resuspended in ice cold homogenization
buffer.
After a further centrifugation (10 min at 4 C and 50,000 g) the cell membranes
were
taken up in ten times their volume made up of binding buffer (50 mM Tris-HCI,
0.5
mM Na-EDTA, 0.01 % ascorbic acid, 10 pM pargylin, pH 7,4) and stored in
portions
(1 ml) at -80 C.
The extract according to the present invention or the alkaloid fractions or
the
polyphenol fractions were dissolved using DMSO in 150 pl binding buffer and
incubated together with 50 pl brain cell membranes (2.5 mg/mI protein) and 50
NI
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radioactive ligand in binding buffer for 45 minutes at room temperature. 3H-
prazosin
(300 pM, specific activity 80 Ci/mmol) was used as the radioligand for the
assay
regarding interactions with a,-adrenoceptors. The unspecific binding was
measured
in the presence of 2 pM phentolamine. 3H-clonidine (1 pM, specific activity
55.5
Ci/mmol) served as the radioligand for the determination of the a2-
adrenoceptor
binding. The assay for the unspecific binding to a2-adrenoceptors was carried
out in
the presence of 10 pM yohimbine. The reaction mixtures were subsequently
filtered
through glass fiber filters (type GF/B) which had been pretreated with
polyethylene
imine (0.2 % in aqua dest.) overnight. After washing the filters twice using 3
ml ice
cold binding buffer each time, the filters were dried for 24 hours at 60 C.
The
determination of the bound radioactivity was carried out after transferring
the filters
into 4 mi scintillization liquid (filter safe, Zinsser-Analytik) in a beta
counter. The
percentage of inhibition of the specific binding of 3H-prazosin to a,-
adrenoceptors or
3H-clonidine to a2-adrenoceptors was calculated as compared to the solvent
control
which had been investigated simultaneously. The determination of the half
maximum
inhibition concentration (IC50 values) was performed by non-linear regression
calculation.
The results of the investigations are summarized in Table 1. It can be seen
that the
extract according to the present invention from the bark of corynanthe
inhibits both
the binding of 3H-prazosin to a,-adrenoceptors and the binding of 3H-clonidine
to a2-
adrenoceptors, this effect being essentially based on the presence of
alkaloids.
Table 1: Inhibition of the binding of 3H-prazosin and 3H-clonidine to al-
adrenoceptors and az-adrenoceptors, respectively
Inhibition of the receptor binding of H-prazosin or H-
clonidine (half maximum inhibition concentration, IC50)
a,-receptor binding a2-receptor binding
Extract according to the
present invention 0.8 pg/mI 2.1 pg/mI
(Example 1)
Polyphenol fraction 10 Ng/mI 10 pg/ml
(Comparative Example 1) no inhibition no inhibition
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Alkaloid fraction 0.05 pg/mI 1 pg/mi
(Comparative Example 2)
Assay for the inhibition of growth factor mediated cell proliferation
The influence of the overall extracts or the extract fractions on the growth
factor
induced cell proliferation was assayed on NIH-3T3 fibroblasts of mice. The
cells were
cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 %
fetal calf serum (FCS), 2 mM glutamine and antibiotic/antimycotic solution.
The
culture medium was regularly replaced twice a week. Four days after the last
subcultivation, adherent cells were detached using trypsine/EDTA from the
bottom of
the cell culture bottle and resuspended in a density of 50,000 cells per ml in
DMEM
supplemented with 0.5 % FCS. Subsequently, the cells were transferred in a
volume
of 200 pl per well into microtiter plates (F-form) and incubated at 37 C for
further 96
h. After replacing the medium (DMEM without FCS) and adding the substances, 10
ng/ml recombinant human platelet-derived growth factor BB (PDGF-BB) was added
60 minutes later. Subsequently, the cells were cultivated again for 24 h at 37
C in an
incubator. Six hours prior to harvesting the cells, 0.5 pCi methyl-3H-
thymidine was
added per well. After the expiry of the incubation period the microtiter
plates were
centrifuged for 5 min at 400 g and the cell supernatants were carefully
pipetted off.
The cells were detached from the bottom using trypsine/EDTA and subsequently
harvested on glass fibre filters (type G-10, ICH-201) using a cell harvester
(Inotech).
The determination of the incorporation of 3H-thymidine in newly synthesized
DNA
was carried out by means of a linear analyzer (LB 2842, Berthold). The
determination of the inhibition of the cell proliferation in the presence of
the extracts
and the extract fractions was carried out in comparison to simultaneously
assayed
solvent controls in each case.
A summary of the results is given in Table 2. It can be seen from these
results that
the cell proliferation inhibiting action of the extracts according to the
present
invention can mainly be attributed to the polyphenol fraction.
Table 2: Inhibition of the PDGF-mediated cell proliferation of NIH3T3
fibroblasts
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half maximum inhibition concentration
Extract according to the
present invention 5.4 Ng/mI
(Example 1)
Polyphenol fraction 3.0 pg/ml
(Comparative Example 1)
Alkaloid fraction 12.3 pg/ml
(Comparative Example 2)
Assay for vasorelaxing properties
For assaying the vasorelaxing effects the influence of extracts according to
the
present invention and extract fractions on the contraction of the isolated
aorta of
male Sprague-Dawley rats (Janvier, Le Genest, France) was examined.
Immediately
after removing, the organs were transferred into Tyrode's solution (in mM:
NaCi
118.2, NaHCO3 24.8, KCI 4.6, CaClz 2.5, MgSO4 1.2, KH2PO4 1.2, glucose 10) and
set free from adhering connective tissue. Then vascular rings having a
thickness of
about 4 mm were prepared. For some experiments the endothelium was removed.
For this purpose the aorta rings were mounted on a steel canula, slightly
pressed
against the steel canula and the innermost vascular layer was subsequently
removed
by turning and simultaneously moving in longitudinal direction. The aorta
rings were
fixed in an organ bath (20 ml; Hugo Sachs, Hugstetten) filled with Tyrode's
solution
using metal hooks. The culture medium (37 C) was permanently gassed with
carbogen (pH 7.4). For experiments regarding the determination of the vascular
relaxation after a precontraction of the organs using phenylephrine (PE), the
following substances were added to the Tyrode's solution: propanolol-HCI (6
pM;
RBI), corticosterone-HCI (6 pL; Sigma) and desipramine (0.6 pM; Sigma). In
tests
regarding the relaxation after stimulation using U-46619 indomethacin (2.8 pM;
Sigma) was added. The organ tension was measured isometrically using a force
transducer (Statham UC2, Hugo Sachs) under a preload of 1.0 g and recorded
using
a 4-channel-writer (Linearcorder, Graphtec). After an equilibration phase of
30 min
four contractions were elicited with PE (0.15 pg/ml, EK 0.74 pM) in intervals
of 15
minutes in order to achieve reproducible organ contractions. Following the
fourth PE
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addition, the test substance was added in an increasing concentration after
reaching
the contraction maximum (cumulative dosage effect). At the end of the
experiment
the endothelium dependency of the vasorelaxation was tested by the application
of
acetylcholine (0.25 pg/ml, EK 1.38 pM).
A basically similar test procedure was complied with also for testing the
relaxing
effect on aorta rings without endothelium. After equilibration for 30 minutes,
three
contractions were elicited with PE (0.15 pg/I, EK 0.74 pM) in intervals of 15
minutes.
After reaching the contraction maximum subsequent to the third addition of PE,
acetylcholine was applied (0.25 pg/mI, EK 1.38 pM) in order to confirm the
complete
removal of the endothelium. After rinsing out the acetylcholine, a PE
contraction
(0.15 pg/mI, EK 0.74 pM) was elicited after 25 minutes and subsequently the
test
substance was added in increasing concentrations.
As described above with respect to PE, assays for a relaxing effect after
inducing a
contraction with U-46619 (0.022 pg/ml, EK 0.063 pM) or KCI (3 mg/ml, EK 40 mM)
was carried out in an identical manner. The relaxing effect of the extracts on
the
agonists was determined as a percentage. IC50 values were determined by a non-
linear regression analysis of concentration-effect-curves using the Prism 3.0
software
(GraphPad Software Inc.).
The results of the experiments are shown in the following table. It is evident
from the
data that the vasorelaxing effect of the extract according to the present
invention
after stimulation with PE is mediated by both the alkaloid containing fraction
and the
alkaloid free fraction. The relaxing effect of the alkaloid free fraction is
completely
dependent on the presence of an intact endothelium. The endothelium-dependent
vasorelaxing effects of the alkaloid free fraction could also be observed
after
precontraction of the vascular rings with U-46619 or KCI-depolarization and
are
obviously based on the increased endothelial release of NO. In contrast, the
effect of
the alkaloid fraction is mainly based on the presence of ingredients having a-
adrenoceptor blocking properties and is, thus, also determinable after
removing the
endothelium.
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Table 3: Assay for vasorelaxing effects
vaso- vaso- vaso- vaso-
relaxation, relaxation, relaxation, relaxation,
PE- PE- U-46619- KCI-
contraction, contraction, contraction, contraction,
endothelium endothelium endothelium endothelium
intact removed intact intact
Extract according to the ED50: ED50: 25 pg/mI: 25 ug/mi:
present invention 0.7 pg/mI 6.3 pg/ml 74 % 61 %
(Example 1)
Polyphenol fraction ED50: 25 pg/ml: ED50: 25 pg/mi:
(Comparative Example 1) 16 pg/ml inactive 16 Ng/mI 25 %
Alkaloid fraction ED50: ED50: 25 pg/mi: 25 Ng/ml:
(Comparative Example 2) 0.22 pg/mI 0.23 pg/ml 15 % inactive
Assay on anti-hypercholesterolaemic effects
The influence of the extract according to the present invention on the plasma
cholesterol level was examined on male NMRI mice (Janvier, Le Genest, France)
with Triton WE1339-induced hypercholesterolaemia. The mice were kept under
standardized environmental conditions (21 C, 60 % relative humidity, 12/12 h
bright/dark) and had free access to drinking water and pelletized feed
(Altromin
1324). Triton-WR1339 (400 mg/kg, Sigma) was dissolved in physiological NaCI
solution and injected into the animals via the caudal vein (10 ml/kg). For an
oral
treatment of the test animals, the extract was taken up in 0.2 % agar
suspension and
administered to the animals 24 h and 1 h prior to the injection of Triton-
WR1339 as
well as 6 h after the injection of Triton-WR1339 via gavage (450 mg/kg in 10
ml/kg).
Animals in the control group were treated with the carrier (0.2 % agar, 10
ml/kg) only.
One hour prior to the injection of Triton-WR1339 and 6 h, 24 h and 48 h after
the
injection of Triton-WR1339, a blood sample (32 pl) was taken from the caudal
vein of
the animals using a heparinized capillary and the cholesterol concentration
was
determined immediately thereafter (Reflotron, Boehringer Mannheim).
Figure 1 shows the influence of the oral treatment with the extract according
to the
present invention (450 mg/kg) on the cholesterol level in mice with Triton
WR1339-
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induced hypercholesterolaemia (# means P < 0.05 as compared to the control (t-
test)). The results of the experiments demonstrate that the treatment with the
extract
according to the present invention obtained in Example 1 leads to a
significant
reduction of the increased cholesterol level.
Assay for analgetic effects
For assaying analgetic effects the formalin test was used in mice. The local
injection
of formalin into the hind foot of mice leads to an increased sensitivity to
pain which
occurs in two temporally separated phases. The first phase is mediated by a
direct
stimulation of the pain receptors due to the release of substance P,
bradykinin and
excitatory amino acids (e.g. glutamine). In the subsequent second phase, an
accumulation of histamine, serotonine and prostaglandins in the tissue occurs,
which
leads to a local inflammation reaction and functional changes in the central
nervous
system. For the experiments, male NMRI mice (Janvier, Le Genest, France)
having a
weight of about 22-26 g were used. The animals were treated orally with the
extracts
according to the present invention or extract fractions. One hour later, 20 pl
of a
3.5 % formalin solution was injected into the left sole of foot. Subsequently,
the
animals were individually placed into wire cages and the number of pain
reactions
(licking the paw) was recorded over a period of 45 min. The pain inhibiting
effect was
determined as compared to a simultaneously tested control group. The animals
in
this group were treated with a carrier medium only (0.2 % agar suspension, 10
mI/kg).
The results in Table 4 demonstrate the potent analgetic effects of corynanthe
extracts which are substantially mediated by the alkaloids contained therein.
Table 4: Assay for analgetic effects
inhibition of the pain reactions
Extract according to the 450 mg/kg
present invention -76 %
(Example 1)
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Polyphenol fraction 450 mg/kg
(Comparative Example 1) -22 %
Alkaloid fraction 100 mg/kg
(Comparative Example 2) -76 %
Assay for antioxidative properties
The autoxidation of lipids is associated with the emission of light. The
determination
of this extraordinarily weak chemiluminescence can be used both for
quantifying
peroxides and for assessing the efficacy of antioxidants. Brain tissue of male
mice
(NMRI; 20 - 30 g; Centre d'Elevage Janvier, Le Genest-Saint Isle, France)
served as
lipid-rich tissue in the present investigations. After its removal the brain
was washed
with ice cold phosphate-buffered physiological saline (PBS, pH 7.4) and set
free from
meninges and remaining blood. The tissue samples were homogenized in 4 times
their volume (v/w) made up of PBS and centrifuged for 10 minutes at 1000 g and
4 C. The supernatants were immediately diluted to three times their volume
with the
same buffer and stored on ice. 250 ial of the diluted supernatant was
transferred into
a test tube and incubated for 10 minutes at 37 C in a 6-channel luminometer
(Multi-
Biolumat LB 9505 C, Berthold, Bad Wildbad). After adding 25 pl of compound II
in
PBS with 2.5 % DMSO, the incubation was continued for a further 10 minutes.
Then
the intensity of the chemiluminescence (CL) was determined for a period of 60
minutes. The percentage of inhibition of the autoxidation was calculated as
compared to a simultaneously tested solvent control (PBS with 2.5 % DMSO). As
can be taken from the results summarized in Table 5, the extract according to
the
present invention exhibits potent antioxidative properties which are primarily
mediated by the polyphenol fraction.
Table 5: Assay for antioxidative properties
inhibition of the lipid peroxidation
IC50 m /ml
Extract according to the present
invention 1151
(Example 1)
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Polyphenol fraction 916
(Comparative Example 1)
Alkaloid fraction 4786
(Comparative Example 2)
Examples
Determination of the total content of phenols according to Folin-Ciocalteu
The determination of the total content of phenols is carried out
photometrically after
reacting with molybdate-wolframate-reagent in analogy to the pharmacopoeia
method for tanning agents (DAB). For this purpose the extract is dissolved in
aqueous ethanol, alkalized with sodium carbonate solution and added with
molybdate-wolframate-reagent. After centrifugation the absorbance of the
supernatant solution is measured against water at 720 nm. The calculation is
based
on epicatechine.
Example 1: Dry extract according to the present invention from the bark of
corynanthe pachyceras
500 g of ground bark of corynanthe pachyceras was stirred twice for one hour
at
60 C using 3.5 kg of 60 % by weight ethanol each time. After filtration over
Seitz
Supra 1500, the combined extract solution were evaporated at about 50 C and
reduced pressure and dried at 50 C in vacuum: 183.8 g (36.8 %).
The extract contains 14.62 % alkaloids (4.75 % corynanthine, 0.81 % a-
yohimbine,
3.86 % corynantheine, 1.91 % dihydrocorynantheine and 3.29 % corynantheidine)
and had a total phenol content of 26.8 % (including 2.66 % epicatechine, 3.05
%
procyanidin B2 and 1.25 % procyanidin Cl).
Comparative Example 1: Polyphenol fraction (alkaloid-free)
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A solution of 445 g dry extract according to Example 1 in 4 kg ethanol (50 %
by
volume) is placed on a column with 3.4 L strongly acidic ion exchanger (Merck
I) and
eluted with ethanol (50 % by volume). 9 L eluate were collected, evaporated at
50 C
under reduced pressure and dried in a drying cabinet at 50 C and 12 mbar:
352.4 g
(79.2 %).
The extract does not contain any alkaloids (corynanthine, a-yohimbine,
corynantheine, dihydrocorynantheine and corynantheidine could not be detected)
and exhibited a total phenol content of 28.4 % (including 2.57 % epicatechine,
2.24
% procyanidin B2 and 0.76 % procyanidin Cl).
Comparative Example 2: Alkaloid fraction
The ion exchanger column from Comparative Example 1 was further eluted using a
mixture of 50 % by volume ethanol and 5 % NH3 solution (having a concentration
of
%). 16 L eluate were collected and evaporated and dried as in Example 2: 46.8
g
(10.5 %).
The extract contains 69.28 % alkaloids (24.01 % corynanthine, 2.25 % a-
yohimbine,
20 19.05 % corynantheine, 9.56 % dihydrochorynanteine and 14.41 %
corynantheidine)
and has a total phenol content of 13.0 % (epicatechine, procyanidin B2 and
procyanidin Cl could not be detected).
Example 2: Tablets
A dry extract form the bark of corynanthe pachyceras (extract according to the
present invention obtained in Example 1) is mixed with adjuvants and pressed
into
tables (tablet core = items 1- 6). The tablets are provided with a coating
made of
hydroxypropyl methyl cellulose (items 7 - 10).
ingredient mg/tablet
dry extract from the bark of
1 corynanthe pachyceras (Example 100.0
1)
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2 microcrystalline cellulose 117.0
3 lactose-monohydrate 58.0
4 croscarmellose 15.0
highly dispersed silicon dioxide 3.0
6 magnesium stearate 6.0
7 hydroxypropylmethyl cellulose 15.0
8 polyethylene glycol 3.0
9 talcum 1.0
titanium dioxide 2.0
22
